Anaplasma phagocytophilum in Ticks Blood-Feeding on Migratory Birds in Sweden.

Migratory birds play a dual role as potential reservoirs of tick-borne pathogens, and potential dispersers of pathogen-containing ticks during their migratory journeys. Ixodes ricinus, a prevalent tick species in Northern and Western Europe, serves as a primary vector for Anaplasma phagocytophilum-a bacterium with implications for human and animal health. There is limited information available regarding A. phagocytophilum in birds. Our investigation focused on A. phagocytophilum prevalence in ticks collected from migratory birds in southeastern Sweden. The identification of ticks involved both molecular analyses for species determination and morphological classification to ascertain the developmental stage. The presence of A. phagocytophilum was determined using real-time PCR. Of the 1115 ticks analyzed from 4601 birds, 0.9% (n = 10), including I. ricinus and Ixodes frontalis, tested positive for A. phagocytophilum. Notably, common blackbirds (Turdus merula) yielded the highest number of A. phagocytophilum-infected ticks. The findings suggest that A. phagocytophilum is present in a small proportion of ticks infesting migratory birds in southeastern Sweden. Consequently, the role of birds as hosts for ticks infected with A. phagocytophilum appears to be low, suggesting that birds seem to play a minor indirect role in the geographic dispersal of A. phagocytophilum.

Coinfection of Babesia and Borrelia in the Tick Ixodes ricinus-A Neglected Public Health Issue in Europe?

Ixodes ricinus nymphs and adults removed from humans, and larvae and nymphs from birds, have been analysed for infection with Babesia species and Borrelia species previously in separately published studies. Here, we use the same data set to explore the coinfection pattern of Babesia and Borrelia species in the ticks. We also provide an overview of the ecology and potential public health importance in Sweden of I. ricinus infected both with zoonotic Babesia and Borrelia species. Among 1952 nymphs and adult ticks removed from humans, 3.1% were PCR-positive for Babesia spp. Of these Babesia-positive ticks, 43% were simultaneously Borrelia-positive. Among 1046 immatures of I. ricinus removed from birds, 2.5% were Babesia-positive, of which 38% were coinfected with Borrelia species. This study shows that in I. ricinus infesting humans or birds in Sweden, potentially zoonotic Babesia protozoa sometimes co-occur with human-pathogenic Borrelia spp. Diagnostic tests for Babesia spp. infection are rarely performed in Europe, and the medical significance of this pathogen in Europe could be underestimated.

Substantial viral and bacterial diversity at the bat-tick interface.

Ticks harbour a high diversity of viruses, bacteria and protozoa. The soft tick Carios vespertilionis (Argasidae) is a common ectoparasite of bats in the Palearctic region and is suspected to be vector and reservoir of viruses and other microbial species in bat populations, some of which may act as zoonotic agents for human disease. The Soprano pipistrelle (Pipistrellus pygmaeus, Vespertilionidae) is widely distributed in Europe, where it can be found inside or close to human habitation. We used meta-transcriptomic sequencing to determine the RNA virome and common microbiota in blood-fed C. vespertilionis ticks collected from a Soprano pipistrelle bat roosting site in south-central Sweden. Our analyses identified 16 viruses from 11 virus families, of which 15 viruses were novel. For the first time in Sweden we identified Issuk-Kul virus, a zoonotic arthropod-borne virus previously associated with outbreaks of acute febrile illness in humans. Probable bat-associated and tick-borne viruses were classified within the families Nairoviridae, Caliciviridae and Hepeviridae, while other invertebrate-associated viruses included members of the Dicistroviridae, Iflaviridae, Nodaviridae, Partitiviridae, Permutotetraviridae, Polycipiviridae and Solemoviridae. Similarly, we found abundant bacteria in C. vespertilionis, including genera with known tick-borne bacteria, such as Coxiella spp. and Rickettsia spp. These findings demonstrate the remarkable diversity of RNA viruses and bacteria present in C. vespertilionis and highlight the importance of bat-associated ectoparasite surveillance as an effective and non-invasive means to track viruses and bacteria circulating in bats and ticks.

First Records of Possibly Human Pathogenic Rickettsia Species in Bat Ticks, Carios vespertilionis, in Sweden.

The Soprano pipistrelle bat, Pipistrellus pygmaeus, is a common species in large parts of Sweden. Many of its natural habitats are near human habitations. This creates opportunities for ticks infesting these bats to encounter humans and possibly transmit zoonotic pathogens by tick bites. The bats are often infested with Carios vespertilionis, a tick species that, in addition to bats, has been recorded to bite humans on occasion. This study aimed to investigate if C. vespertilionis acts as a reservoir for Anaplasma phagocytophilum, Neoehrlichia mikurensis, Tick-borne encephalitis virus, and species of Babesia and Rickettsia and to improve currently used conventional PCR protocols for molecular species determination of Rickettsia spp. Ninety-two C. vespertilionis ticks were collected from underneath a bat-box harbouring P. pygmaeus. Pathogen-specific PCR assays showed that 58.4% were positive for Rickettsia spp. and negative for the other pathogens analysed. Phylogenetic analyses indicate that the species belong to R. parkeri, R. conorii, R. slovaca, R. sibirica subsp. mongolotimonae, R. rickettsii, and a hitherto uncultured Rickettsia sp. Several of these species are considered pathogenic to humans. Given the ecology and behaviour of C. vespertilionis, it may be a vector of these rickettsiae among bats and occasionally humans. To determine the Rickettsia species with certainty, and to determine if C. vespertilionis may be a reservoir and vector of the Rickettsia spp., further studies are needed.

Co-Occurrence of Francisella, Spotted Fever Group Rickettsia, and Midichloria in Avian-Associated Hyalomma rufipes.

The migratory behavior of wild birds contributes to the geographical spread of ticks and their microorganisms. In this study, we aimed to investigate the dispersal and co-occurrence of Francisella and spotted fever group Rickettsia (SFGR) in ticks infesting birds migrating northward in the African-Western Palaearctic region (AWPR). Birds were trapped with mist nests across the Mediterranean basin during the 2014 and 2015 spring migration. In total, 575 ticks were collected from 244 birds. We screened the ticks for the species Francisella tularensis, the genus Francisella, and SFGR by microfluidic real-time PCR. Confirmatory analyses and metagenomic sequencing were performed on tick samples that putatively tested positive for F. tularensis during initial screenings. Hyalomma rufipes was the most common tick species and had a high prevalence of Francisella, including co-occurrence of Francisella and SFGR. Metagenomic analysis of total DNA extracted from two H. rufipes confirmed the presence of Francisella, Rickettsia, and Midichloria. Average nucleotide identity and phylogenetic inference indicated the highest identity of the metagenome-assembled genomes to a Francisella-like endosymbiont (FLE), Rickettsia aeschlimannii, and Midichloria mitochondrii. The results of this study suggest that (i) FLE- and SFGR-containing ticks are dispersed by northbound migratory birds in the AWPR, (ii) H. rufipes likely is not involved in transmission of F. tularensis in the AWPR, and (iii) a dual endosymbiosis of FLEs and Midichloria may support some of the nutritional requirements of H. rufipes.

Association between guilds of birds in the African-Western Palaearctic region and the tick species Hyalomma rufipes, one of the main vectors of Crimean-Congo hemorrhagic fever virus.

INTRODUCTION: The ecology of the vertebrate host contributes to the geographical range expansion of ticks. In this study, we investigated which tick taxa that infest and are dispersed by birds along African-Western Palaearctic flyways during northward migration, and whether bird ecology was associated with tick taxa. MATERIALS AND METHODS: Ticks were collected from birds trapped at bird observatories in Spain, Italy, Greece, and Israel during the spring migration of 2014 and 2015, using mist nets. The tick-infested bird species were classified into guilds, using different combinations of the variables: migration distance, wintering region, foraging behaviour, and winter habitat. Ticks were molecularly determined to genus and species level by sequencing fragments of the 12S ribosomal DNA (rDNA) gene and by phylogenetic inference, using the Maximum Likelihood algorithm. Data were analysed using descriptive measures, graphs, Chi2 tests, the Tukey-Kramer test, and a parametric linear model (generalized linear model) in order to analyse and adjust for characteristics in the bird guilds and their relationship to collected tick taxa. RESULTS: Most (84.2%) of the 10,209 trapped birds were long-distance migrants, of which 2.4% were infested by ticks. The most common tick species was Hyalomma rufipes (77.7%; 447/575), a known vector and reservoir of Crimean-Congo hemorrhagic fever virus. Bird guilds containing only long-distance migrants with wintering areas in Africa were associated with the tick species H. rufipes (p < 0.0001). Furthermore, bird winter habitat was associated with H. rufipes (p = 0.003); with bird species overwintering in open habitat (p = 0.014) and wetlands (p = 0.046) having significantly more H. rufipes as compared to birds with a winter habitat comprising forest and shrubs (p = 0.82). CONCLUSIONS: With climate change, the likelihood of establishment of permanent Hyalomma populations in central and northern Europe is increasing. Thus, surveillance programs for monitoring the risk of introduction and establishment of H. rufipes in the Western-Palaearctic should be established. Our study suggests that migratory bird species wintering in African open habitats and wetlands are good candidates for monitoring potential introduction.

First Record of a Suspected Human-Pathogenic Borrelia Species in Populations of the Bat Tick Carios vespertilionis in Sweden.

The bat tick Cariosvespertilionis has been reported from Sweden to occasionally feed on humans resulting in disease symptoms. The aim of this study was to investigate C. vespertilionis as a potential vector and reservoir of Borrelia species. In 2015 and 2018 in south-central Sweden, C. vespertilionis ticks were collected from a wooden bat box harboring Soprano pipistrelle bats, Pipistrellus pygmaeus. In addition, one C. vespertilionis tick found inside a house in southern Sweden in 2019 was collected. Ticks were screened for Borrelia spp. using a genus-specific quantitative PCR assay. The Borrelia species of the positive specimens were determined by conventional PCR followed by DNA sequencing and phylogenetic analyses. A total of 24% (22 of 92) of the analyzed C. vespertilionis ticks were Borrelia-positive. Phylogenetic analyses indicate that the bacteria belong to the relapsing fever group of borreliae; some of them appear to be identical with Borrelia sp. CPB1, a spirochete only found twice before-in the United Kingdom and in France. Our results also indicate a temporal and spatial distribution of this Borrelia species. Since C. vespertilionis occasionally bites humans, and since it exhibits a high prevalence of Borrelia bacteria, it is possible that it presents a risk of human disease. Further studies are needed to characterize Borrelia sp. CPB1 to determine if it is human-pathogenic and to determine if C. vespertilionis is a vector and/or reservoir of this agent.

Three Babesia species in Ixodes ricinus ticks from migratory birds in Sweden.

BACKGROUND: Migratory birds can cross geographical and environmental barriers and are thereby able to facilitate transmission of tick-borne pathogens both as carriers of infected ticks and as reservoirs of pathogenic microorganisms. Ixodes ricinus is one of the most abundant tick species in the Northern Hemisphere and a main vector of several Babesia species, some which pose a potential threat to human and animal health. At present only two cases of overt babesiosis in humans have so far been reported in Sweden. To better understand the potential role of birds as disseminators of zoonotic Babesia protozoan parasites, we investigated the presence of Babesia species in ticks removed from migratory birds. METHODS: Ticks were collected from birds captured at Ottenby Bird Observatory, south-eastern Sweden, from March to November 2009. Ticks were molecularly identified to species, and morphologically to developmental stage, and the presence of Babesia protozoan parasites was determined by real-time PCR. RESULTS: In total, 4601 migratory birds of 65 species were examined for tick infestation. Ticks removed from these birds have previously been investigated for the presence of Borrelia bacteria and the tick-borne encephalitis virus. In the present study, a total of 1102 ticks were available for molecular analysis of Babesia protozoan parasites. We found that 2.4% of the ticks examined, all I. ricinus, were positive for mammal-associated Babesia species. Out of all Babesia-positive samples, Babesia venatorum was the most prevalent (58%) species, followed by Babesia microti (38%) and Babesia capreoli (4.0%). B. venatorum and B. capreoli were detected in I. ricinus larvae, whereas B. microti was only present in I. ricinus nymphs. This supports the view that the two first-mentioned species are vertically (transovarially) transmitted in the tick population, in contrast to B. microti. The largest number of Babesia-infected ticks was removed from the common redstart (Phoenicurus phoenicurus) and European robin (Erithacus rubecula). CONCLUSIONS: This study reveals that Babesia protozoan parasites are present in ticks infesting migratory birds in south-eastern Sweden, which could potentially lead to the dissemination of these tick-borne microorganisms into new areas, thus posing a threat to humans and other mammals.

Migratory birds as disseminators of ticks and the tick-borne pathogens Borrelia bacteria and tick-borne encephalitis (TBE) virus: a seasonal study at Ottenby Bird Observatory in South-eastern Sweden.

BACKGROUND: Birds can act as reservoirs of tick-borne pathogens and can also disperse pathogen-containing ticks to both nearby and remote localities. The aims of this study were to estimate tick infestation patterns on migratory birds and the prevalence of different Borrelia species and tick-borne encephalitis virus (TBEV) in ticks removed from birds in south-eastern Sweden. METHODS: Ticks were collected from resident and migratory birds captured at the Ottenby Bird Observatory, Öland, Sweden, from March to November 2009. Ticks were molecularly identified to species, and morphologically to developmental stage, and the presence of Borrelia bacteria and TBEV was determined by quantitative real-time PCR. RESULTS: A total of 1339 ticks in the genera Haemaphysalis, Hyalomma, and Ixodes was recorded of which I. ricinus was the most abundant species. Important tick hosts were the European robin (Erithacus rubecula), Blackbird (Turdus merula), Tree pipit (Anthus trivialis), Eurasian wren (Troglodytes troglodytes), Common redstart (Phoenicurus phoenicurus), Willow warbler (Phylloscopus trochilus), and Common whitethroat (Sylvia communis). Borrelia bacteria were detected in 25% (285/1,124) of the detached ticks available for analysis. Seven Borrelia species (B. afzelii, B. burgdorferi (s.s.), B. garinii, B. lusitaniae, B. turdi, B. valaisiana, and B. miyamotoi) were identified. B. turdi was recorded for the first time in ticks in Sweden. The number of Borrelia cells per tick ranged from 2.0 × 100 to 7.0 × 105. B. miyamotoi-containing ticks contained a significantly higher median number of Borrelia cells than B. burgdorferi (s.l.)-containing ticks. B. garinii and B. miyamotoi were the most prevalent Borrelia species in tick larvae. Larvae of I. ricinus with B. garinii were removed from seven bird species, particularly S. communis and A. trivialis, which may suggest that the larvae had contracted the Borrelia bacteria from or via these birds. Also, a high percentage of tick larvae containing B. miyamotoi was removed from E. rubecula. All ticks were negative for TBEV. CONCLUSIONS: The results corroborate the view that the contributions of birds to human disease are substantial, particularly as blood hosts for ticks and for their short-, medium-, and long-distance dispersal. Moreover, several ground-foraging bird species appear to be important for the maintenance and dispersal of Borrelia species. The absence of TBEV in the ticks conforms to other similar studies.

A divergent Anaplasma phagocytophilum variant in an Ixodes tick from a migratory bird; Mediterranean basin.

Anaplasma phagocytophilum (AP) has vast geographical and host ranges and causes disease in humans and domesticated animals. We investigated the role of northward migratory birds in the dispersal of tick-borne AP in the African-Western Palearctic. Ticks were collected from northward migratory birds trapped during spring migration of 2010 at two localities in the central Mediterranean Sea. AP DNA was detected by PCR (gltA and 16S rRNA) and variant determination was performed using ankA sequences. In total, 358 ticks were collected. One of 19 ticks determined as Ixodes was confirmed positive for AP DNA. The tick was collected from a woodchat shrike (Lanius senator senator) trapped in Greece, and molecularly determined to belong to the I. ricinus complex and sharing highest (95%) 16S RNA sequence identity to I. gibbosus. The ankA AP sequence exhibited highest similarity to sequences from rodents and shrews (82%) and ruminants (80%). Phylogenetic analyses placed it convincingly outside other clades, suggesting that it represents a novel AP variant. The divergent Ixodes species harboring a novel AP variant could either indicate an enzootic cycle involving co-evolution with birds, or dissemination from other regions by avian migration. None of the 331 Hyalomma marginatum sensu lato ticks, all immature stages, were positive for AP DNA, lending no evidence for the involvement of Hyalomma ticks transported by birds in the ecology of AP.

First records of adult Hyalomma marginatum and H. rufipes ticks (Acari: Ixodidae) in Sweden.

From July 2018 to January 2019 we recorded 41 specimens of adult Hyalomma ticks, which had been found on horses, cattle or humans in 14 Swedish provinces. In 20 cases we received tick specimens, which were identified morphologically as adults of H. marginatum (n = 11) or H. rufipes (n = 9). These are the first documented records in Sweden of adults of H. marginatum and H. rufipes. Molecular tests for Crimean-Congo haemorrhagic fever virus and piroplasms (Babesia spp. and Theileria spp.) proved negative; 12 out of 20 tested specimens were positive for rickettsiae (R. aeschlimannii was identified in 11 of the ticks). All ticks originated from people or animals that had not been abroad during the previous two months. These data suggest (i) that the adult Hyalomma ticks originated from immature ticks, which had been brought from the south by migratory birds arriving in Sweden during spring or early summer; and that (ii) due to the exceptionally warm summer of 2018 these immature ticks had been able to develop to the adult stage in the summer and/or autumn of the same year. The rapidly changing climate most likely now permits these two Hyalomma species to develop to the adult, reproductive stage in northern Europe. There is consequently a need to revise the risk maps on the potential geographic occurrence of relevant tick species and related tick-borne pathogens in Sweden and in the neighbouring countries.

First records of tick-borne pathogens in populations of the taiga tick Ixodes persulcatus in Sweden.

BACKGROUND: The common tick Ixodes ricinus and the taiga tick I. persulcatus are the main tick vectors of Borrelia spirochaetes, TBE virus (TBEV) and of several other zoonotic pathogens in the western and eastern areas, respectively of the Palaearctic region. Recently, populations of the taiga tick were, for the first time, detected in northern Sweden. This prompted us to investigate if they harbour human pathogens. METHODS: A total of 276 I. persulcatus ticks (136 males, 126 females and 14 nymphs) and one I. ricinus nymph was collected by the cloth-dragging method in northern Sweden in July-August 2015 and May-July 2016. In addition, 8 males and 10 females of I. persulcatus were collected from two dogs (16 and 2 ticks, respectively) in two of the localities. All ticks were microscopically and molecularly identified to developmental stage and species and screened for B. burgdorferi (sensu lato), B. miyamotoi, Anaplasma phagocytophilum, Rickettsia spp., Neoehrlichia mikurensis, Babesia spp. and TBEV using real-time PCR followed by species identification by sequencing the PCR-products of conventional PCR assays. RESULTS: Of the ticks collected by the cloth-dragging method, 55% (152/277) were positive for Borrelia. There was no significant difference between the proportions of Borrelia-infected nymphs (33%, 5/15) and Borrelia-infected adult ticks (56%, 147/262), and no significant difference between the proportions of Borrelia-infected males (54%, 74/136) and Borrelia-infected females (58%, 73/126). Three different Borrelia species were identified. Borrelia afzelii was the predominant species and detected in 46% of all Borrelia-infected ticks followed by B. garinii, 35%, B. valaisiana, 1%, and mixed infections of different Borrelia species, 1%; 17% of all Borrelia-infections were untypeable. One I. persulcatus female contained Rickettsia helvetica, and one nymph contained Rickettsia sp. Of the 277 ticks analysed, all were negative for A. phagocytophilum, Babesia spp., Borrelia miyamotoi, N. mikurensis and TBEV. The ticks collected from the two dogs were negative for all pathogens examined except for Borrelia spp., that was detected in 5 out of 16 ticks removed from one of the dogs. CONCLUSIONS: To our knowledge, this is the first time that I. persulcatus from Sweden has been analysed for the presence of tick-borne pathogens. The examined tick populations had a low diversity of tick-borne pathogens but a high prevalence of B. burgdorferi (s.l.).

The importance of wildlife in the ecology and epidemiology of the TBE virus in Sweden: incidence of human TBE correlates with abundance of deer and hares.

BACKGROUND: Tick-borne encephalitis (TBE) is one tick-transmitted disease where the human incidence has increased in some European regions during the last two decades. We aim to find the most important factors causing the increasing incidence of human TBE in Sweden. Based on a review of published data we presume that certain temperature-related variables and the population densities of transmission hosts, i.e. small mammals, and of primary tick maintenance hosts, i.e. cervids and lagomorphs, of the TBE virus vector Ixodes ricinus, are among the potentially most important factors affecting the TBE incidence. Therefore, we compare hunting data of the major tick maintenance hosts and two of their important predators, and four climatic variables with the annual numbers of human cases of neuroinvasive TBE. Data for six Swedish regions where human TBE incidence is high or has recently increased are examined by a time-series analysis. Results from the six regions are combined using a meta-analytical method. RESULTS: With a one-year time lag, the roe deer (Capreolus capreolus), red deer (Cervus elaphus), mountain hare (Lepus timidus) and European hare (Lepus europaeus) showed positive covariance; the Eurasian elk (moose, Alces alces) and fallow deer (Dama dama) negative covariance; whereas the wild boar (Sus scrofa), lynx (Lynx lynx), red fox (Vulpes vulpes) and the four climate parameters showed no significant covariance with TBE incidence. All game species combined showed positive covariance. CONCLUSIONS: The epidemiology of TBE varies with time and geography and depends on numerous factors, i.a. climate, virus genotypes, and densities of vectors, tick maintenance hosts and transmission hosts. This study suggests that the increased availability of deer to I. ricinus over large areas of potential tick habitats in southern Sweden increased the density and range of I. ricinus and created new TBEV foci, which resulted in increased incidence of human TBE. New foci may be established by TBE virus-infected birds, or by birds or migrating mammals infested with TBEV-infected ticks. Generally, persistence of TBE virus foci appears to require presence of transmission-competent small mammals, especially mice (Apodemus spp.) or bank voles (Myodes glareolus).

Alkhurma Hemorrhagic Fever Virus RNA in Hyalomma rufipes Ticks Infesting Migratory Birds, Europe and Asia Minor.

Alkhurma hemorrhagic fever virus RNA was detected in immature Hyalomma rufipes ticks infesting northward migratory birds caught in the North Mediterranean Basin. This finding suggests a role for birds in the ecology of the Alkhurma hemorrhagic fever virus and a potential mechanism for dissemination to novel regions. Increased surveillance is warranted.

First evidence of established populations of the taiga tick Ixodes persulcatus (Acari: Ixodidae) in Sweden.

BACKGROUND: The tick species Ixodes ricinus and I. persulcatus are of exceptional medical importance in the western and eastern parts, respectively, of the Palaearctic region. In Russia and Finland the range of I. persulcatus has recently increased. In Finland the first records of I. persulcatus are from 2004. The apparent expansion of its range in Finland prompted us to investigate if I. persulcatus also occurs in Sweden. METHODS: Dog owners and hunters in the coastal areas of northern Sweden provided information about localities where ticks could be present. In May-August 2015 we used the cloth-dragging method in 36 localities potentially harbouring ticks in the Bothnian Bay area, province Norrbotten (NB) of northern Sweden. Further to the south in the provinces Västerbotten (VB) and Uppland (UP) eight localities were similarly investigated. RESULTS: Ixodes persulcatus was detected in 9 of 36 field localities in the Bothnian Bay area. Nymphs, adult males and adult females (n = 46 ticks) of I. persulcatus were present mainly in Alnus incana - Sorbus aucuparia - Picea abies - Pinus sylvestris vegetation communities on islands in the Bothnian Bay. Some of these I. persulcatus populations seem to be the most northerly populations so far recorded of this species. Dog owners asserted that their dogs became tick-infested on these islands for the first time 7-8 years ago. Moose (Alces alces), hares (Lepus timidus), domestic dogs (Canis lupus familiaris) and ground-feeding birds are the most likely carriers dispersing I. persulcatus in this area. All ticks (n = 124) from the more southern provinces of VB and UP were identified as I. ricinus. CONCLUSIONS: The geographical range of the taiga tick has recently expanded into northern Sweden. Increased information about prophylactic, anti-tick measures should be directed to people living in or visiting the coastal areas and islands of the Baltic Bay.

Candidatus Neoehrlichia mikurensis in Ticks from Migrating Birds in Sweden.

Candidatus Neoehrlichia mikurensis (CNM; family Anaplasmataceae) was recently recognized as a potential tick-borne human pathogen. The presence of CNM in mammals, in host-seeking Ixodes ticks and in ticks attached to mammals and birds has been reported recently. We investigated the presence of CNM in ornithophagous ticks from migrating birds. A total of 1,150 ticks (582 nymphs, 548 larvae, 18 undetermined ticks and two adult females) collected from 5,365 birds captured in south-eastern Sweden was screened for CNM by molecular methods. The birds represented 65 different species, of which 35 species were infested with one or more ticks. Based on a combination of morphological and molecular species identification, the majority of the ticks were identified as Ixodes ricinus. Samples were initially screened by real-time PCR targeting the CNM 16S rRNA gene, and confirmed by a second real-time PCR targeting the groEL gene. For positive samples, a 1260 base pair fragment of the 16S rRNA gene was sequenced. Based upon bacterial gene sequence identification, 2.1% (24/1150) of the analysed samples were CNM-positive. Twenty-two out of 24 CNM-positive ticks were molecularly identified as I. ricinus nymphs, and the remaining two were identified as I. ricinus based on morphology. The overall CNM prevalence in I. ricinus nymphs was 4.2%. None of the 548 tested larvae was positive. CNM-positive ticks were collected from 10 different bird species. The highest CNM-prevalences were recorded in nymphs collected from common redpoll (Carduelis flammea, 3/7), thrush nightingale (Luscinia luscinia, 2/29) and dunnock (Prunella modularis, 1/17). The 16S rRNA sequences obtained in this study were all identical to each other and to three previously reported European strains, two of which were obtained from humans. It is concluded that ornithophagous ticks may be infected with CNM and that birds most likely can disperse CNM-infected ticks over large geographical areas.

Spotted fever Rickettsia species in Hyalomma and Ixodes ticks infesting migratory birds in the European Mediterranean area.

BACKGROUND: A few billion birds migrate annually between their breeding grounds in Europe and their wintering grounds in Africa. Many bird species are tick-infested, and as a result of their innate migratory behavior, they contribute significantly to the geographic distribution of pathogens, including spotted fever rickettsiae. The aim of the present study was to characterize, in samples from two consecutive years, the potential role of migrant birds captured in Europe as disseminators of Rickettsia-infected ticks. METHODS: Ticks were collected from a total of 14,789 birds during their seasonal migration northwards in spring 2009 and 2010 at bird observatories on two Mediterranean islands: Capri and Antikythira. All ticks were subjected to RNA extraction followed by cDNA synthesis and individually assayed with a real-time PCR targeting the citrate synthase (gltA) gene. For species identification of Rickettsia, multiple genes were sequenced. RESULTS: Three hundred and ninety-eight (2.7%) of all captured birds were tick-infested; some birds carried more than one tick. A total number of 734 ticks were analysed of which 353 ± 1 (48%) were Rickettsia-positive; 96% were infected with Rickettsia aeschlimannii and 4% with Rickettsia africae or unidentified Rickettsia species. The predominant tick taxon, Hyalomma marginatum sensu lato constituted 90% (n = 658) of the ticks collected. The remaining ticks were Ixodes frontalis, Amblyomma sp., Haemaphysalis sp., Rhipicephalus sp. and unidentified ixodids. Most ticks were nymphs (66%) followed by larvae (27%) and adult female ticks (0.5%). The majority (65%) of ticks was engorged and nearly all ticks contained visible blood. CONCLUSIONS: Migratory birds appear to have a great impact on the dissemination of Rickettsia-infected ticks, some of which may originate from distant locations. The potential ecological, medical and veterinary implications of such Rickettsia infections need further examination.

Prevalence of tick-borne encephalitis virus in Ixodes ricinus ticks in northern Europe with particular reference to Southern Sweden.

BACKGROUND: In northern Europe, the tick-borne encephalitis virus (TBEV) of the European subtype is usually transmitted to humans by the common tick Ixodes ricinus. The aims of the present study are (i) to obtain up-to-date information on the TBEV prevalence in host-seeking I. ricinus in southern and central Sweden; (ii) to compile and review all relevant published records on the prevalence of TBEV in ticks in northern Europe; and (iii) to analyse and try to explain how the TBE virus can be maintained in natural foci despite an apparently low TBEV infection prevalence in the vector population. METHODS: To estimate the mean minimum infection rate (MIR) of TBEV in I. ricinus in northern Europe (i.e. Denmark, Norway, Sweden and Finland) we reviewed all published TBEV prevalence data for host-seeking I. ricinus collected during 1958-2011. Moreover, we collected 2,074 nymphs and 906 adults of I. ricinus from 29 localities in Sweden during 2008. These ticks were screened for TBEV by RT-PCR. RESULTS: The MIR for TBEV in nymphal and adult I. ricinus was 0.28% for northern Europe and 0.23% for southern Sweden. The infection prevalence of TBEV was significantly lower in nymphs (0.10%) than in adult ticks (0.55%). At a well-known TBEV-endemic locality, Torö island south-east of Stockholm, the TBEV prevalence (MIR) was 0.51% in nymphs and 4.48% in adults of I. ricinus. CONCLUSIONS: If the ratio of nymphs to adult ticks in the TBEV-analysed sample differs from that in the I. ricinus population in the field, the MIR obtained will not necessarily reflect the TBEV prevalence in the field. The relatively low TBEV prevalence in the potential vector population recorded in most studies may partly be due to: (i) inclusion of uninfected ticks from the 'uninfected areas' surrounding the TBEV endemic foci; (ii) inclusion of an unrepresentative, too large proportion of immature ticks, compared to adult ticks, in the analysed tick pools; and (iii) shortcomings in the laboratory techniques used to detect the virus that may be present in a very low concentration or undetectable state in ticks which have not recently fed.

On the potential roles of ticks and migrating birds in the ecology of West Nile virus.

BACKGROUND: Mosquitoes are the primary vectors of West Nile virus (WNV). Ticks have, however, been suggested to be potential reservoirs of WNV. In order to investigate their role in the spread of the virus, ticks, which had been collected from birds migrating northwards from Africa to Europe, were analyzed for the potential presence of WNV-RNA. METHODS: On the Mediterranean islands Capri and Antikythira a total of 14,824 birds were captured and investigated from which 747 ticks were collected. RESULTS AND CONCLUSION: Most of the identified ticks (93%) were nymphs and larvae of Hyalomma marginatum sensu lato, most of which were or appear to be Hyalomma rufipes. Of these ticks 729 were individually screened for WNV-RNA. None of the ticks was found to be WNV positive. Thus, there was no evidence that Hyalomma marginatum s.l. ticks play a role in the spread of WNV from Africa to Europe.